Genetically-engineered mhc molecules

ABSTRACT

The invention provides DNA molecules encoding a chimeric polypeptide comprising (a) a component of a MHC molecule capable of association on a cell surface with an endogenous MHC molecule component of the same class, and (b) an intracellular region of a signal transduction element capable of activating T cells. Component (a) may be a monomorphic component and is preferably beta 2-microglobulin, or a polymorphic class I or class II component. The signal transduction element (b) capable of activating T cells may be a component of T-cell receptor CD3, preferably the CD3 zeta (zeta) polypeptide, a B cell receptor polypeptide or an Fc receptor polypeptide. Immune cells such as a CTLs expressing said chimeric MHC molecules specifically eliminate or inactivate harmful T cells and are useful for treating graft rejection and autoimmune diseases.

FIELD OF THE INVENTION

[0001] The present invention is in the field of Immunology and relatesto an immunotherapeutic approach designed to specifically eliminate orinactivate undesired harmful T cells. In particular, the inventionrelates to DNA constructs encoding chimeric molecules comprising a MHCcomponent, and a signal transduction element component, and to cellstransfected with such DNA molecules that are useful in the preventionand/or treatment of T-cell mediated disorders and conditions such asgraft rejection and autoimmune diseases.

[0002] ABBREVIATIONS: APC—antigen-presenting cell; β₂m—β₂ microglobulin;CTL—cytotoxic T lymphocyte; EAE—experimental autoimmuneencephalomyelitis; Ha—hemagglutinin; HLA—human leukocyte antigen (=humanMHC); IB—insulin B chain; IDDM—insulin-dependent diabetes mellitus;Ig—immunoglobulin; ITAM—immunoreceptor tyro sine-based activation motif;mAb—monoclonal antibody; MHC—major histocompatibility complex;MS—multiple sclerosis; NP—nucleoprotein; RT-PCR—reversetranscriptase-polymerase chain reaction; TCR—T-cell receptor; T_(H)—Thelper cells.

BACKGROUND OF THE INVENTION

[0003] Lymphocytes are the main cells of the immune system responsiblefor acquired immunity and the immunologic characteristics of diversity,specificity, memory and self/nonself recognition. Lymphocytes can bebroadly divided into B cells, characterized by the presence ofmembrane-bound immunoglobulin (antibody) molecules which serve asreceptors for, and can bind, soluble antigens; and T cells,characterized by the presence of membrane-bound receptor molecules (TCR,for T-cell receptor), which recognize and bind antigen only when theantigen is associated with a self-molecule encoded by genes within themajor histocompatibility complex (MHC). The MHC is referred to as theH-2 complex in mice and as the HLA (for human leukocyte antigen) complexin humans.

[0004] T-cell recognition of antigen is the basis of the adaptive immuneresponse, which is the ability of the immune system to selectivelyrecognize, neutralize and obliterate infectious pathogens orpathological cells. Effector T cells generated in response to antigenare responsible for cell-mediated immunity. All T-cell subpopulationsexpress the TCR, but they can be distinguished by the presence of one orthe other of two membrane glycoprotein molecules on their surfaces(surface antigens), CD4 or CD8, and their role in the immune response.Thus, two major subpopulations of T cells can be characterized: the CD4⁺or helper T cells (T_(H)), which facilitate the activities of other celltypes, and the CD8⁺ or cytotoxic T lymphocytes (CTL), which can directlykill abnormal or infected cells.

[0005] CD4⁺ T cells are further divided into T_(H)1 inflammatory Tcells, which secrete various cytokines (T_(H)1 response) that activatemainly T cytotoxic cells and macrophages responsible for theintracellular destruction of phagocytosed microorganisms, and T_(H)2 Tcells, which secrete various cytokines (T_(H)2 response) that activate Bcells to produce antibodies. CD8⁺ T cells generally function as CTLs.Another T-cell related lymphocyte subset is the natural killer cells(NK), which are large, granulated lymphocytes displaying cytotoxicactivity against a wide variety of tumor and other abnormal cells.

[0006] T-cell recognition is mediated by direct interaction of the TCRwith an antigenic peptide displayed by a MHC product on anantigen-presenting cell (APC). As a result of specific interactionbetween TCR of a T lymphocyte that has not yet interacted with antigen(naive or unprimed cell) with MHC molecules displaying antigenicpeptides on a professional APC (e.g. dendritic cells, macrophages, Bcells), the T cell is activated and it proliferates and eventuallydifferentiates to produce effector cells, which function as T_(H) cellsor CTLs to eliminate the antigen, and memory cells, which areresponsible for the life-long immunity observed for many pathogens.

[0007] In mammals, there are two types of MHC molecules: MHC class Imolecules, which are present on almost all nucleated cells in the bodyand are recognized by CD8⁺ cells, and MHC class II molecules, which aremainly expressed on professional APCs of the immune system and arerecognized by CD4⁺ cells. Thus, CD8⁺ T cells, which generally functionas CTLs, are said to be class I restricted, and CD4⁺ T cells, whichgenerally function as T_(H) cells, are said to be class II restricted.

[0008] Class I MHC molecules are composed of a polymorphic heavy chain(α) non-covalently associated with a monomorphic (in humans) non-MHCencoded light chain protein of about 12 kDa, termed β₂ microglobulin(β₂m). The heavy α chain is a polymorphic transmembrane glycoprotein ofabout 45 kDa consisting of 3 extracellular domains, each containingabout 90 amino acids (α₁ at the N-terminus, α₂ and α₃), a transmembraneregion of about 40 amino acids and a cytoplasmic tail of about 30 aminoacids. The α₁ and α₂ domains, the membrane distal domains, form thepeptide-binding groove or cleft having a sufficient size to bind apeptide of 8-10 amino acids, whereas the α₃ domain is proximal to theplasma membrane. β₂m has a single immunoglobulin (Ig)-like domain, notanchored to the plasma membrane, and interacts mainly with the α₃ chain,which also possesses a characteristic Ig fold. In humans, there arethree α chain genes, called HLA-A, HLA-B and HLA-C, for each of whichmultiple alleles have been identified. In mice, there are three α chaingenes, called H-2K, H-2D and H-2L. CD8⁺ T cells recognize peptides inthe context of MHC class I molecules.

[0009] Class II MHC molecules contain two different polypeptide chains,a 33-kD α chain and a 28-kDa β chain, which associate by noncovalentinteractions. Like class I MHC molecules, class II MHC molecules aremembrane-bound glycoproteins that contain extracellular domains, atransmembrane segment and a cytoplasmic tail. Each chain in thesenoncovalent heterodimeric complexes contains two extracellular domains:α₁ and α₂ domains and β₁ and β₂ domains. The membrane-distal domain of aclass II molecule is composed of the α1 and β1 domains and forms thepeptide-binding groove or cleft having a sufficient size to bind apeptide, which is typically of 13-18 amino acids. The membrane-proximaldomains, α2 and β2, have structural similarities to Ig constant (C)domains. Three pairs of class II α and β chain genes exist in humans,known as HLA-DR, HLA-DP and HLA-DQ. The highest level of polymorphism isdocumented for HLA-DR. This polymorphism is solely contributed by theDRβ chain, as DRα is monomorphic. In mice, the pairs of genes are calledH-2IA and H-2IE. CD4⁺ T_(H) cells recognize peptides in the context ofclass II MHC molecules.

[0010] All T cells bind their specific MHC::peptide antigen viaclone-specific or clonotypic TCR molecules. TCRs are disulfide-linkedheterodimeric transmembrane proteins made of α and β chains. TheN-terminal variable (V) domains of these chains, which together form theantigen-binding site, are similar to those of Ig variable (V) chains,whereas the membrane-anchored C-terminal domains are analogous to Igconstant (C) domains.

[0011] On the T-cell membrane, the clonotypic TCR associatesnon-covalently with CD3, forming the TCR-CD3 membrane complex. CD3, thesignal transduction element of the TCRs, is composed of a group ofinvariant proteins called gamma (γ), delta (δ), epsilon (ε), zeta (ζ)and eta (η) chains. The γ, δ and ε chains are structurally-related, eachcontaining an Ig-like extracellular constant domain followed by atransmembrane region and a cytoplasmic domain of more than 40 aminoacids. The ζ and η chains have a distinctly different structure: bothhave a very short extracellular region of only 9 amino acids, atransmembrane region and a long cytoplasmic tail containing 113 and 115amino acids in the ζ and η chains, respectively. The invariant proteinchains in the CD3 complex associate to form noncovalent heterodimers ofthe ε chain with a γ chain (εγ) or with a δ chain (εδ) or of the ζ and ηchain (ζη), or a disulfide-linked homodimer of two ζ chains (ζζ). About90% of the CD3 complex incorporate the ζζ homodimer.

[0012] The cytoplasmic regions of the CD3 chains contain a motifdesignated the immunoreceptor tyrosine-based activation motif (ITAM).This motif is found in a number of other: receptors including theIg-α/Ig-β heterodimer of the B-cell receptor complex and Fc receptorsfor IgE and IgG. The ITAM sites have been shown to associate withcytoplasmic tyrosine kinases and to participate in signal transductionfollowing TCR-mediated triggering. In CD3, the γ, δ and ε chains eachcontain a single copy of ITAM, whereas the ζ and η chains harbour threeITAMs in their long cytoplasmic regions. Indeed, the ζ and η chains havebeen ascribed a major role in T cell activation signal transductionpathway (Howe and Weiss, 1995; Sherman and Chattopadhyay, 1993).

[0013] In mammals, T cell maturation occurs in the thymus. Duringmaturation, a distinct mechanism operates to ensure positive selection,namely that αβ TCRs expressed in a given individual cell will recognizeand bind to self-MHC molecules, as well as negative selection, namely toeliminate those T cells bearing high affinity TCRs which may interactwith self-MHC molecules alone or self-antigens plus self-MHC molecules,that would pose the threat of an autoimmune response if they matured.Hence, the mature T-cell repertoire can provide an adequate defenseagainst pathogens, while avoiding response against self-antigens.However, peripheral tissue antigens often fail to be adequatelypresented in the thymus, and potentially self-reactive T cells dosucceed to exit the thymus, while downstream mechanisms, which induceperipheral tolerance are not always sufficient to inactivate thesepotentially harmful T cells. In addition, no mechanism has evolved toeliminate or inactivate T cells capable of reacting against foreign MHCallelic products, referred to as alloreactive cells. As a result, theaction of harmful T cells may inflict severe damage and may beassociated with life-threatening situations commonly associated withT-cell mediated diseases and conditions such as autoimmune diseases andtransplantation, and with responses against innocuous foreign antigens,resulting in hypersensitivity reactions such as allergy and asthma.

[0014] Autoimmune disorders are characterized by reactivity of theimmune system to an endogenous antigen, with consequent injury totissues. More than 80 chronic autoimmune diseases have beencharacterized that affect virtually almost every organ system in thebody. The most common autoimmune diseases are insulin-dependent diabetesmellitus (IDDM), multiple sclerosis (MS), systemic lupus erythematosus(SLE), rheumatoid arthritis, several forms of anemia (pernicious,aplastic, hemolytic), thyroiditis, and uveitis. Autoimmune disorders arefar more prevalent in women and are among the top 10 causes of death ofyoung and middle-aged women in the U.S.A.

[0015] Autoimmune diseases result from sustained adaptive immuneresponses mounted against innocuous self-antigens. The effectormechanisms that eventually cause tissue damage and disease are mostlikely those that take part in normal adaptive responses, and includeproduction of specific antibodies, generation of immune complexes,inflammatory and cytotoxic T cells and activated macrophages.

[0016] The role of T cells in autoimmune diseases has been extensivelystudied in MS, a chronic inflammatory disease of the central nervoussystem (CNS) and its rodent model experimental autoimmuneencephalomyelitis (EAE), (for review, see Hafler and Weiner, 1995). InMS, activated CD4⁺ T cells found in the central nervous system (CNS)display specificity to a number of abundant CNS proteins, includingmyelin basic protein (MBP), proteolipid protein (PLP), myelinoligodendrocyte-associated protein (MOG) and S-100. Susceptibility to MSis associated with the HLA-DR2 haplotype approximately 50-70% of MSpatients carry the DR2 allele, which is found in only 20-30% of normalindividuals. This association has enabled, for example, theidentification of immunodominant MBP peptides using panels ofHLA-DR2-restricted T cell clones (Wucherpfennig, et al., 1991).

[0017] A large bulk of data has also been accumulated in IDDM along withits non-obese diabetic (NOD) mouse model. In IDDM, CD4⁺ T cellautoantigens include insulin, GAD (glutamic acid decarboxylase) 65,GAD67, hsp (heat-shock protein) 65, and ICA (islet-cell antigen) 69(Roep, 1996). Recently, a study of the NOD mouse system, which employeda novel screening strategy, has enabled the identification of an insulinB-chain peptide as the first CD8⁺ T cell epitope in an autoimmunedisease (Wong, et al., 1999).

[0018] In summary, a limited number of peptides derived from proteinsinvolved in autoimmune diseases are associated with the onset of thedisease. The immune responses to self-antigens are maintained by thepersistent activation of self-reactive T cells. Removal of T cellpopulations that are associated with the autoimmune response should leadto prevention and/or cure of the disease. This model was demonstrated inthe NOD mice, where the removal of T-cell populations that recognizeproinsulin II, prevented the onset of IDDM (French, et al., 1997).

[0019] Allograft rejection typically results from an overwhelmingadaptive immune response against foreign organ or tissue. It is themajor risk factor in organ transplantation and is the cause ofpost-transplantation complications. A major complication associated withbone marrow (BM) transplantation, known as graft-versus-host (GVH)reaction or graft-versus-host disease (GVHD), occurs in at least half ofpatients when grafted donor lymphocytes, injected into an allogeneicrecipient whose immune system is compromised, begin to attack the hosttissue, and the host's compromised state prevents an immune responseagainst the graft Alloreactivity is complex and involves many cell typesas well as inflammatory factors. It is largely mediated by both CD8⁺(CTL) and CD4⁺ (T_(H)) T cells (for review, see Douillard et al., 1999;Hernandez-Fuentes et al., 1999; Pattison and Krensky, 1997).

[0020] Allograft rejection results from proper recognition of foreignMHC and activation of the adaptive immune system and is carried out bydirect or indirect pathways. The direct pathway, where T-cell receptorsdirectly recognize intact allo-MHC with or without bound peptides on thesurface of target cells, apparently accounts for most of the CTLfunction. The indirect pathway, where T-cell receptors recognize MHCallopeptides after processing and presentation, leads to the activationof T helper cells. These cells provide the necessary signals for thegrowth and maturation of effector CTLs and B cells leading to rejection(Sherman and Chattopadhyay, 1993; Watschinger, 1995).

[0021] The actual role of specific peptides in direct allorecognition isambiguous. Some studies demonstrate that allorecognition ispeptide-independent (Mullbacher et al., 1999; Smith et al., 1997), whileothers imply that specific peptides do contribute to allorecognition(Wang, et al., 1998). Allorecognition may, therefore, comprisepeptide-independent, peptide-dependent or peptide-specific interactions.

[0022] Ideally, treatment of autoimmune diseases should reduce only theautoimmune response while leaving the rest of the immune system intact.In the absence of such treatments, current therapies of autoimmunediseases include non-immune-specific treatments via a broad spectrum ofimmunosuppressive drugs such as corticosteroids, cyclosporine A,methotrexate and tacrolimus (FK506). However, these agents have severeside effects such as “generalized immunosuppression” and toxicity.

[0023] Successful transplant engraftment and effective treatment ofautoimmune diseases would be greatly facilitated by tolerizing,inactivating or eliminating harmful, or potentially harmful, T-cells. Inboth cases, specificity (or at least high selectivity) of the therapy ismandatory if severe side effects are to be minimized. For maximalefficacy and broad applicability, anti-T cell protocols are to targetthe whole repertoire of (allo or auto)specific T cell clones, and to actindependently of HLA identity. Indeed, design and evaluation of specifictreatment strategies have been the objective of numerous studies in boththese fields. Among the approaches that were explored, are anti-T cellidiotypic manipulation (Bluestone et al., 1986), reduction of antigenpresentation by the graft (Carpenter et al., 1976; Faustman, 1995) andsuppression of T cell precursors by veto cells (Miller et al., 1988;Reich-Zeliger et al., 2000; Thomas et al., 1991).

[0024] Identification of potential autoantigens and characterization ofautoantigen-specific T cell clones, mainly in MS (or EAE) and IDDM, haveprompted active research aimed at developing specific immunotherapies toautoimmune diseases. Experimental therapeutic approaches include T-cellvaccination with autoreactive T cells (Ben-Nun and Cohen, 1981) or theirTCR peptides (Howell et al., 1989; Vandenbark et al. 1991), toleranceinduction by oral antigens (reviewed in Hafler and Weiner, 1995),peptide blockade of MHC molecules by specific peptides associated withthe disease or their analogues (Elias et al., 1991; Windhagen et al.,1995), and monoclonal antibody treatment However, wide clinicalapplicability of these approaches still has to be demonstrated.Undoubtedly, novel solutions are to be constantly pursued so thataccumulating knowledge and understanding of the immune system areeventually reduced to wide medical practice.

[0025] Specific direction of an immune intervention procedure againstthe pool of auto-or allo-specific T-cell clones demands a well-definedstructural or functional common denominator which can serve as anidentification tag for this pool. For example, the concept ofvaccination with TCR peptides or whole autoreactive T cells (Howell, etal., 1989; Vandenbark, et al., 1991) is based on the observation thatencephalitogenic T cells in EAE utilize only a restricted set ofgerm-line TCR Vα and Vβ genes, hence, their TCR amino acid sequencesconstitute a potential (although not fully specific) marker. However,autoreactive T cell clones isolated in MS are more promiscuous in theirTCR usage and are thus less accessible to this strategy. The trivialcommon denominator of the whole panel of T cell clones recognizing agiven MHC::peptide ligand is the ligand itself, irrelevant of TCR geneproducts of those clones. The problem is how this understanding can beexploited so that those cells can be specifically targeted for therapy.A novel mechanism, devised to trigger a potent reaction against those Tcells following their specific interaction with the ligand is a likelysolution. This can only be achieved if the ligand is engineered to belinked to an adequate effector function, activated upon its engagementwith specific TCRs.

[0026] In recent years, the inventors (Eshhar et al., 1993; Gross etal., 1995; Gross et al., 1989; Hwu, et al., 1993) and others (reviewedin Gross and Eshhar, 1992), have demonstrated that genetic engineeringenables redirecting T cell recognition at will via chimeric activationreceptors. This was accomplished by replacement of the ligand-bindingdomain of a T-cell receptor, with binding domains derived from otherreceptor molecules. Other studies have shown that reciprocalsubstitution of transmembrane and intracellular domains of surfacereceptors with those of different molecules involved in T-cellactivation signal transduction leads to a corresponding change in thepattern of response to the same signal. The transmembrane andcytoplasmic regions of the CD3 ζ chain or Ig Fc receptor γ chain havebeen frequently used in such experiments, proving most powerful in thisregard (e,g. Eshhar et al., 1993; Irving and Weiss, 1991; Letourneur andKlausner, 1991; Romeo and Seed, 1991).

[0027] A number of publications disclose chimeric receptors comprising aCD3 ζ chain and an extracellular binding domain. For example, Seed etal., in U.S. Pat. No. 5,843,728, disclose chimeric receptors comprisingthe extracellular domain of CD4 fused to an intracellular portion of aTCR CD3 ζ or η chain, a B-cell receptor polypeptide or an Fc receptorpolypeptide. T lymphocytes expressing these chimeras would recognize andkill cells expressing HIV gp120.

[0028] U.S. Pat. No. 5,855,740 and U.S. Pat. No. 5,834,266 disclosechimeras comprising an intracellular CD3 ζ chain and an extracellulardomain capable of specifically binding to at least one ligand. U.S. Pat.No. 5,359,046 discloses chimeras optionally containing an intracellularCD3 ζ chain fused to extracellular domains derived from CD4, CD8, Ig orsingle-chain antibody. U.S. Pat. No. 5,712,149 discloses chimericcostimulatory receptors whose intracellular domain is derived from CD2or CD28 and may, in addition, comprise a CD3 ζ chain domain.

SUMMARY OF THE INVENTION

[0029] It is an object of the present invention to provide a novelconcept and means for specific removal, by elimination or inactivation,of undesired T cells such as autoreactive T cells causing autoimmunediseases and alloreactive T cells causing transplant rejection or GVHD.

[0030] The present invention is based on the principle that MHC productscan be genetically engineered into T-cell activation molecules and that,when these MHC components are monomorphic, the engineered molecules canact independently of MHC identity. Thus, a MHC component geneticallyengrafted with intracellular structural elements responsible fortransduction of T cell activation signals, may be functionally expressedon the membrane of T cells, particularly cytotoxic T lymphocytes (CTLs),together with relevant antigenic peptides, and these modified T cells(effector cells) will target specific, harmful T cells bearing theantigen receptors (target T cells) and will cause lysis or inactivationof the undesired T cells.

[0031] The present invention thus relates, in one aspect, to a DNAmolecule encoding a chimeric polypeptide comprising (a) a component of aMHC molecule capable of association on a cell surface with an endogenousMHC molecule component of the same class, and (b) an intracellularregion of a signal transduction element capable of activating T cellsand, optionally, an antigenic peptide related to an autoimmune diseaselinked to said chimeric polypeptide by a peptide linker.

[0032] According to the present invention, once MHC components aresupplemented with a signal transduction element capable of activating Tcells such as the transmembranal and cytoplasmic regions of the CD3 ζchain, they will be endowed with the capacity to transduce T-cellactivation signal. If expressed by CTLs, these chimeric MHC moleculescan function as traps: T cells interacting with them (while occupiedwith antigenic peptides) are destined to be lysed. In other words, sucha manipulation is expected to provoke an ‘inverted’ response: fromligand to TCR.

[0033] In further aspects, the invention relates to a vector comprisinga DNA molecule of the invention and to cells, particularly T cells suchas CTLs, expressing a chimeric polypeptide encoded by the DNA moleculeof the invention.

[0034] In still a further aspect, the invention relates to methods forprevention and/or treatment of T-cell mediated diseases or conditionssuch as autoimmune diseases and transplant rejection, which comprisesadministering to a patient in need thereof immune cells, particularlyCTLs, that express a chimeric polypeptide encoded by the DNA molecule ofthe invention.

BRIEF DESCRIPTION OF THE FIGURES

[0035]FIG. 1 shows schematic drawings of native class I and class II MHCmolecules and of chimeric MHC molecules linked to a zeta (ζ) polypeptideaccording to the invention. Left panel—MHC class I., native (left) andchimeric (right). β₂m—β₂ microglobulin; α1, α2, α3—heavy chain domains;bridge—a short peptide connecting β₂m to the cell membrane;arrow—activation signal. Right panel—MHC class II, native (left) andchimeric (right). α1, α2—α chain domains; β1, β2—β chain domains.

[0036]FIG. 2 depicts a chimeric human β₂m/ζ genetic construct of theinvention cloned downstream of SRα promoter of the pBJ1-Neo expressionvector. pr—promoter; lead—leader peptide of human β₂m; br—HLA-A2-derivedpeptide bridge; tm+cyt—transmembrane and cytoplasmic domains of CD3 ζ.Numbers and directions of PCR primers used for cloning are indicated.

[0037]FIG. 3 shows the DNA sequences of the chimeric hβ₂m/ζ gene and itsseparate components. Upper panel: DNA sequence of the coding region ofthe human β₂m gene (from GenBank Accession AF072097). Middle panel: Theregion encoding mouse CD3 ζ chain transmembranal and cytoplasmic domains(from GenBank Accession M19729). Lower panel: The sequence of thechimeric hβ₂m/ζ gene, joined via the XhoI site, in clone 21-2 (Example2). Restriction sites used for cloning are underlined: tctaga—XbaI;ctcgag—XhoI; gaattc—EcoRI. atg initiation codons, taa stop codons andthe HLA-A2 bridge coding sequence are shaded.

[0038]FIG. 4 shows flow cytometry analysis of surface expression of hβ₂mon MD45 hybridoma parental cells and two hβ₂m/ζ transfectants, 29 and 49(Example 2). Pale line—staining with an anti-hβ2m mAb. Dark line—controlstaining with an irrelevant antibody.

[0039]FIG. 5 shows IL-2 production after stimulation of chimeric hβ₂m/ζtransfectants with an immobilized anti-H-2K^(k) mAb. IL-2 was monitoredwith a CTL-L bioassay, which was developed via an MTT colorimetric assay(see “Materials and Methods”). MD45—parental hybridoma cells; 29 and49—primary transfectants; 29-2 and 49-10—respective sub-clones. TCGFcontrol reflects maximal CTL-L growth.

[0040] FIGS. 6A-6B show components for the preparation of a chimericantigenic peptide/hβ₂m/ζ construct and a representation thereof. FIG. 6Ashows transformation of a native class I MHC molecule (left) into achimeric molecule with a ζ chain via a bridge peptide (middle) and to afurther chimeric molecule in which an antigenic peptide is linked to theN-terminus of the chimeric hβ₂m/ζ molecule (right). FIG. 6B depicts achimeric antigenic peptide/hβ₂m/ζ construct designed to load antigenicpeptides onto the construct of FIG. 6A. pr—the SRα promoter; lead—leaderpeptide of hβ₂m; p—antigenic peptide; li—peptide linker; br—HLA-A2peptide bridge; tm+cyt —transmembranal and cytosplasmic domains. Theconstruct was cloned downstream of SRα promoter of the pBJ1-Neoexpression vector. Numbers and directions of PCR primers used forcloning are indicated. Primers 6086, 6087 and 6338 are specific to theNP, Ha and IB peptides, respectively. (Example 4).

[0041]FIG. 7 shows the DNA sequences encoding the three chimericantigenic peptide/hβ₂m/ζ polypeptides described in Example 4. Upperpanel: DNA sequence of the coding region of the chimeric nucleoprotein(NP) peptide/hβ₂m/ζ gene from clone 406-20. Middle panel: The part ofthe coding region of the chimeric hemagglutinin (Ha) peptide/hβ₂m/ζ genein clone 4074, from its 5′ end till the BamHI site in the peptidelinker-encoding segment. From this site till its 3′ end this gene isidentical to 406-20. Lower panel: The part of the coding region of thechimeric insulin B chain (IB) peptide/hβ₂m/ζ gene in clone 408-9, fromits 5′ end till the BamHI site in the peptide linker-encoding segment.From this site till its 3′ end this gene is identical to 406-20. Allsequences are identical to that of clone 21-2 (see FIG. 3) starting 24nucleotides downstream of the underlined BamHI site. Restriction sitesused for cloning are underlined: tctaga—XbaI; ggattc—BamHI; ctcgag—XhoI;gaattc—EcoRI. atg initiation codons, taa stop codons, the HLA-A2 bridgeand the three antigenic peptide coding sequences are shaded.

[0042]FIG. 8 is a graph showing the results of β-galactosidase (β-Gal)enzymatic assay of the dose-dependent response of clone 425-68 to serialdilutions of an immobilized anti-H-2K^(k) mAb (Example 6).Concentrations of the mAb which was applied into the experiment well aregiven in the x axis on a logarithmic scale. Results are in OD₄₀₅.

[0043]FIG. 9 shows β-Gal production by clones 425-44 (expressingNP/hβ₂m/ζ and NF-AT-LacZ) and 427-24 (expressing Ha/hβm/ζ and NF-ATLacZ) after incubation with the two target cells (Example 6). Theresults are the summary of 15 independent experiments. Results are givenin μ units of β-Gal according to a standard curve produced in eachexperiment with commercial β-Gal (Promega, Wis., USA). Statisticalsignificance was assessed by ANOVA.

[0044] FIGS. 10A-10B show a schematic representation of the chimericDRα/ζ gene, clone 23-2 (Example 7), wherein numbers and directions ofPCR primers used for cloning are indicated, and the DNA sequence of thechimeric DRα/ζ gene 23-2 and its separate components, respectively. InFIG. 10B: Upper panel—DNA sequence of the coding region of the humanHLA-DRα (DRA*0101 gene in GenBank Accession J00194). Lower panel: DNAsequence of the chimeric DRα/ζ gene, joined via the XbaI site.Restriction sites used for cloning are underlined: ctcgag—XhoI;tctaga—XbaI; gaattc—EcoRI. atg initiation codons, taa stop codons andthe one junctional nucleotide from each of the two genes are shaded

DETAILED DESCRIPTION OF THE INVENTION

[0045] Specific direction of an immunointervention procedure against thepool of auto- or allo-specific T cells in autoimmune disease or inallograft rejection, respectively, demands a well-defined structural orfunctional common denominator which can serve as an identification tagfor this pool.

[0046] The idea underlying the present invention is that the trivialcommon denominator of the whole panel of T cells is to recognize a givenMHC::peptide ligand, irrespective of TCR gene products of these cells.The present invention provides a new mechanism devised to trigger apotent reaction against harmful T cells by following their specificinteraction with the MHC::peptide ligand. This can only be achieved ifthe ligand is engineered to be linked to an adequate effector function,activated upon its engagement with specific TCRs.

[0047] The present invention demonstrates the feasibility of creatinggenetically-engineered DNA molecules encoding a chimeric polypeptidecomprising a MHC component and a signal transduction element component.When introduced into cells, particularly T cells, this construct willendow the cells with the capacity to transduce T-cell activation signal.Thus, T cells expressing these chimeric polypeptides together withrelevant antigenic peptide, are rendered functional against otherspecific T cells which are restricted by the modified MHC products.

[0048] The present invention relates to a DNA molecule encoding achimeric polypeptide comprising (a) a component of a MHC moleculecapable of association on a cell surface with an endogenous MHC moleculecomponent of the same class, and (b) an intracellular region of a signaltransduction element capable of activating T cells.

[0049] In one preferred embodiment of the invention, the MHC component(a) is a monomorphic component such as the non-MHC encoded humanβ₂-microglobulin (β₂m) molecule or the cc chain of a HLA-DR molecule.

[0050] In one most preferred embodiment, component (a) is monomorphicβ₂m, the class I. MHC light chain, which is capable of association on acell surface with an endogenous class I heavy chain HLA molecule. Theβ₂m molecule is linked to component (b) by a bridge peptide having about10-15 amino acid residues. Preferably, this bridge peptide has asequence comprised within the membrane-proximal sequence of a class Iheavy chain HLA molecule. In a preferred embodiment, this bridge peptidehas 13 amino acid residues comprised within the extracellularmembrane-proximal sequence of the class I heavy chain HLA-A2 molecule,and has the sequence:

[0051] Leu Arg Trp Glu Pro Ser Ser Gln Pro Thr Ile Pro Ile

[0052] In another preferred embodiment, component (a) is the monomorphicHLA-DRα chain, which is capable of association on a cell surface with anendogenous HLA-DRβ chain.

[0053] In another embodiment, the MHC component (a) is a polymorphiccomponent selected from: (i) an α chain of class I HLA-A, HLA-B or HLA-Cmolecule, which is capable of association on a cell surface withendogenous β₂m; (ii) an α chain of class II HLA-DP or HLA-DQ molecule,which is capable of association on a cell surface with endogenousHLA-DPβ or HLA-DQβ chain; or (iii) a polymorphic β chain of class IIHLA-DP, BLA-DR or HLA-DQ molecule, which is capable of association on acell surface with an endogenous HLA-DPα, HLA-DRα or HLA-DQα chain.

[0054] Component (b) of the chimeric activation receptor of theinvention comprises the intracellular region of a suitable signaltransduction element capable of activating T cells such as, but notbeing limited to, a component of T-cell receptor CD3 such as the zeta(ζ) or eta (η) polypeptide, a B cell receptor polypeptide or an Fcreceptor polypeptide. As mentioned in the Background sectionhereinbefore, the cytoplasmic regions of the CD3 chains contain a motifdesignated the immunoreceptor tyrosine-based activation motif (ITAM),which has been shown to associate with cytoplasmic tyrosine kinases andto participate in signal transduction following TCR-mediated triggering.This motif is found in a number of other receptors including theIg-α/Ig-β heterodimer of the B-cell receptor complex and Fc receptorsfor IgE and IgG, and three copies of it are found in the longcytoplasmic domains of the the ζ and η chains.

[0055] In a preferred embodiment, component (b) of the chimeric moleculecomprises the transmembranal and cytoplasmic regions of the human CD3 ζpolypeptide.

[0056] In another embodiment, component (b) comprises the transmembranaland cytoplasmic regions of a B-cell receptor polypeptide such as theIg-α or Igβ chain, the cytoplasmic tails in both being long enough tointeract with intracellular signaling molecules. In a furtherembodiment, component (b) comprises the transmembranal and cytoplasmicregions of Fc receptor polypeptides such as FcεRI, FcγRI or FcγRIIIchains. FcγRI, a high-affinity receptor expressed on the surface of mastcells and basophils, contains four polypeptide chains: an α and β chainand two identical disulfide-linked γ chains that extend a considerabledistance into the cytoplasm and each has an ITAM motif FcγRI, or CD64,is the high affinity receptor for IgG, expressed mainly on macrophages,neutrophils, eosinophils and dendritic cells. It comprises an α chainand two disulfide-linked γ chains. This structure is also typical toFaγRIII, or CD16, which is the low affinity receptor for IgG, found onNK cells, eosinophils, macrophages, neutrophils and mast cells. CD3 ζchain is found instead of the γ chain in a fraction of FcγRIII.

[0057] In an additional embodiment, the invention provides a DNAmolecule encoding for a chimeric polypeptide as described above, whereinsaid chimeric polypeptide further comprises an antigenic peptide relatedto an autoimmune disease, said antigenic peptide being linked tocomponent (a) of said chimeric polypeptide by a peptide linker. Theantigenic peptide will typically have 8-10 amino acid residues whencomponent (a) is class I β₂m or α chain, and will typically have 13-18amino acid residues when component (a) is class II α or β chain.

[0058] The DNA molecule of the invention is constructed from components(a) and (b) under a suitable promoter by cloning in a suitable vector byDNA manipulation techniques well-known in the art. For example, cDNAsegments encoding components (a) and (b) are cloned by reversetranscriptase-polymerase chain reaction (RT-PCR) using mRNA fromappropriate cells, with suitable primers that contain appropriaterestriction sites for insertion of the PCR products into a suitableexpression vector. The resulting vector containing the DNA encoding thechimeric polypeptide is then used for transfection of the desired cells.

[0059] In another aspect, the invention relates to an expression vectorcomprising a DNA molecule of the invention. Any expression vectorsuitable for transfection or infection of mammalian cells and in whichcells the vectors are capable of directing the regulated expression ofcloned genes into recombinant polypeptides, can be used according to theinvention. Since the invention relates to the expression of the chimericpolypeptide in human cells, these vectors are preferably viral vectorssuch as retroviral or adenovirus vectors.

[0060] In another aspect, the present invention provides a cell whichexpresses a chimeric polypeptide comprising (a) a component of a MHCmolecule capable of association on a cell surface with an endogenous MHCmolecule component of the same class, and (b) an intracellular region ofa signal transduction element capable of activating T cells. The cellsare preferably immune cells selected from T helper cells (CD4⁺), naturalkiller (NK) cells and, most preferably, T cytotoxic cells (CTL, CD8⁺),capable of recognizing and binding to harmful T cells and causing theirelimination or inactivation.

[0061] Immune cells which express a chimeric polypeptide containing onlycomponents (a) and (b) without any additional peptide, are capable ofbinding to, and eliminating, alloreactive cells causing transplantrejection.

[0062] Immune cells which express a chimeric polypeptide containingcomponents (a) and (b) and one or more additional peptides related to acertain autoimmune disease, will be able to bind to, and eliminate,autoreactive cells causing said autoimmune disease. For this purpose,the peptide (or peptides) can also be exogenously supplied by incubationof cells expressing a chimeric polypeptide containing only components(a) and (b) with one or more antigenic peptides or one or more proteinsassociated with the autoimmune disease to be treated. The peptide, orpeptides, can also be processed and presented by the immune cellfollowing the introduction into the cell of a gene encoding one peptideor a number of peptides, from the same protein or from differentproteins associated with the disease.

[0063] The invention further provides a method for prevention and/ortreatment of graft rejection, which comprises administering to a patientin need thereof at suitable times T cells which express a chimericpolypeptide comprising (a) a component of a MHC molecule capable ofassociation on a cell surface with an endogenous MHC molecule componentof the same class, and (b) an intracellular region of a signaltransduction element capable of activating T cells. For the treatment ofgraft-versus-host disease (GVHD), the cells administered are autologousT cells, whereas for the treatment of host-versus-graft reaction, thecells administered are donor T cells.

[0064] The invention still further provides a method for the preventionand/or treatment of an autoimmune disease which comprises administeringto a patient in need thereof autologous T cells which express a chimericpolypeptide comprising an antigenic peptide related to said autoimmunedisease linked by a peptide linker to a component of a MHC moleculecapable of association on a cell surface with an endogenous MHC moleculecomponent of the same class, followed by an intracellular region of asignal transduction element capable of activating T cells.

[0065] The invention yet further provides a method for the preventionand/or treatment of an autoimmune disease which comprises administeringto a patient in need thereof autologous T cells which express a chimericpolypeptide comprising (a) a component of a MHC molecule capable ofassociation on a cell surface with an endogenous MHC molecule componentof the same class, and (b) an intracellular region of a signaltransduction element capable of activating T cells, and wherein one ormore antigenic peptides or proteins related to said autoimmune diseaseare exogenously loaded in the groove of the MHC complex formed by theassociation of component (a) with the endogenous MHC molecule component,or processed and presented by the MHC complex following internalizationof exogenous polypeptides or expression of an introduced gene encodingthe antigenic peptide or protein.

[0066] The working hypothesis underlying this invention is that once MHCcomponents expressed in T cells (=effector cells) are supplemented withT cell signaling domains from T-cell activation molecules such as theCD3 ζ and η chains, these chimeric MHC-signal transduction elementmolecule will be capable of triggering T-cell activation uponencountering specific TCRs on other T cells (=target cells). On thesurface of the effector T cell, these chimeric MHC components areexpected to pair with their complementary, native MHC products and allownormal antigen presentation.

[0067] The MHC components in these chimeric polypeptides can either bemonomorphic such as β₂m (for class I) or HLA-DRα (for class II), orpolymorphic, such as all class I heavy α chains and other HLA class II αor β chains. The signal transducing element of the chimeric polypeptidecan be, for example, the ζ chain of the CD3 complex or the γ chain of anIg Fc receptor complex. When the effector cell is a CTL, activation isexpected to lead to target T cell lysis via Fas signaling or cytotoxinrelease.

[0068] This concept lays the grounds for a novel immunotherapeuticapproach designed to direct genetically-modified CTL against harmful Tcells, resulting in specific elimination or inactivation of the latter.This approach may be applicable to transplantation and autoimmunediseases (and to other disorders, such as allergy or asthma), targetingonly specific alloreactive or autoreactive CD4⁺ and CD8⁺ T cells,respectively.

[0069] A special characteristic of the invention is its universalitywhen monomorphic MHC components are employed, meaning that the chimericgenes can be applied in a universal manner, namely, irrespective of HLAalleles.

[0070] In transplantation protocols, donor CTL will be transducedex-vivo with the DNA molecule encoding the chimeric MHC polypeptide,selected for expression of the introduced gene, expanded throughcontinuous activation and administered to the recipient in proper timingand amount during the transplantation procedure. In the class Isituation, these cells will be able to -lyse allospecific recipient CTL,with potential coverage of the entire HLA class I allelic spectrum dueto β₂m monomorphism. Since alloreactivity is often not peptide-specific,the majority of recipient alloreactive cells will become targets.Recipient CTL specific to peptides presented by the graft but not bydonor CTL are expected to escape recognition, but are likely toconstitute only a minor fraction of the alloreactive cell population.Activated human T cells, including CD8⁺ cells, express class II moleculeand can function as professional antigen presenting cells (Barnaba, etal., 1994) Chimeric HLA class II-transduced, activated donor CTL shouldtherefore recognize allospecific recipient CD4⁺ cells. A recent reportof a clinical trial (Bonini, et al., 1997) demonstrates that geneticallymodified donor T cells can indeed function in the recipient. Suicidegene-mediated elimination of such cells following allogeneic BMtransplantation could control host-versus-graft reaction in leukemicpatients.

[0071] For (GVHD prevention, recipient T cells isolated and propagatedprior to treatment, can be similarly transduced ex-vivo with chimericclass I and class II genes. Following their re-infusion into thepatient, these cells are expected to act specifically against recipientanti-donor T cells.

[0072] In autoimmune diseases associated, for example, with HLA-DR,patient's CD8⁺ T cells are transduced ex-vivo with a chimeric DRα gene,selected, activated and cultured with the autoantigen proteins orpeptides. Upon re-infusion into the patient, these cells are expected toact specifically against DR-restricted autoreactive cells.

[0073] Another possible result of the therapeutic protocols suggestedherein is the triggering of an anti-TCR immune response, similar inessence to those obtained following vaccination with TCR peptides orwith whole autoreactive T cells (Howell, et al., 1989;Vandenbark, etal., 1991), and directed specifically against the pathogenic cells. Suchan outcome may be mediated through local cytokine release by theengineered effector T cells.

[0074] In addition to CTL lytic machinery, genetic coupling of MHCchimeras to other effector functions in either CD4⁺ or CD8⁺ cells, canbe pursued. For example, in MS, it may be favorable to instruct them tomediate secretion of cytokines which suppress inflammatory T cells, suchas TGF-β, upon specific interaction with their target T cells.

[0075] The invention will now be illustrated by the followingnon-limiting Examples.

EXAMPLES

[0076] Materials and Methods

[0077] Materials: Monoclonal antibodies (mAbs) to mouse H-2K^(k) andH-2K^(d) were purchased from PharMingen (San Diego, Calif., USA).Another mAb to mouse H-2 K^(k) (from rat) was purchased from Zymed (SanFrancisco, Calif., USA). MAb against human β₂m, polyclonal goatanti-mouse IgG (FAB specific)-FITC conjugated, puromycin,β-Galactosidase, methyl tetrazolium (MTT) and concanavalin A (ConA) wereall purchased from Sigma (St Louis, Mo., USA). G418 Sulphate was fromLife Technologies (Glasgow, UK).

[0078] Cell lines: MD45 is a CTL hybridoma of BALB/c mice, allospecificto H-2b (Kaufmann et al., 1981). Cells were cultured in DMEM,supplemented with 10% heat-inactivated fetal calf serum (F[-FCS), 2 mML-Glutamine, 1 mM sodium pyruvate, Pen-Strep Solution (penicillin 10,000units/ml, streptomycin, 10 mg/ml at 1:1000 dilution), all fromBiological Industries (Beit Haemek, Israel). HK9.5-24 and HK-8.3-5H3 areCTL hybridomas of BALB/1 origin (Stryhn et al., 1994). Cells werecultured in RPMI 1640 (Biological Industries, Beit Haemek, Israel)supplemented with 10% HI-FCS, 2 mM L-glutamine, 50 μM β-mercaptoethanol(β-ME), 10 mM HEPES buffer and Pen-Strep Solution at 1:1000 dilution.CTL-L cell line was cultured in RPMI 1640 medium supplemented with 5%HI-FCS, 10 mM Hepes, 50 μM β-ME, 2 mM L-glutamine, Pen-Strep solution at1:1000 dilution supplemented with 5% TCGF (from rat serum followingpolyclonal T cell activation).

[0079] DNA transfection: An 0.8 ml of 1.5×10⁶ cell/ml (MD45 orsubsequent clones) were mixed in 4 mm sterile electroporation cuvette(ECU-104, EquiBio, Ashford, UK) with 10 μg DNA of the constructedplasmid and 5 μg DNA of the selection plasmid and placed on ice.Transfection was performed by electroporation using Easyject Pluselectroporation unit (EquiBio, Ashford, UK) at 350V, 750 μF. Cells wereresuspended in fresh medium and cultured for 24-48 hours in 96-wellplates prior to addition of the selecting drug (2 mg/ml G418 or 1 μg/mlpuromycin). Resistant clones were first expanded in 24-well plates.

[0080] FACS analysis: 10⁶ cells were washed with phosphate-bufferedsaline (PBS) containing 0.02% sodium azide and incubated for 30 minuteson ice with 100 μl of the anti-human β₂m mAb (Sigma) at 10 μg/ml or thesame concentration of a control antibody. Cells were then washed andincubated on ice with 100 μl of 1:100 dilution of goat anti-mouse IgG(FAB specific)-FITC conjugated polyclonal antibody (Sigma) for 30minutes. Cells were washed and resuspended in PBS and analyzed by a FlowCytometer (Becton Dickinson).

[0081] Cell stimulation assay: Cells at 5×10⁵ cells/ml were incubatedovernight in 24-well plates in the presence of Con A (usually at 10μg/ml), anti-mouse K^(k) or K^(d) mAb (usually at 5 μg/ml, immobilizedovernight and washed 3 times in PBS) or with target cells at 5×10⁵cells/ml. Total volume was 1 ml.

[0082] β-Galactosidase enzymatic assay: 20-24 hours post-stimulation,cells were harvested in 1.5 ml Eppendorf tubes and centrifuged at 7000rpm for 2 min at room temperature. Supernatant was collected for an IL-2production assay. The pellet was washed 3 times in fresh PBS and assayedfor β-galactosidase (β-Gal) using β-Galactosidase Enzyme Assay SystemKit (Promega, Wis., USA) according to the manufacturer's instructions.The assay was developed in 96-well plates and was read with SLT SpectraELISA Reader (SLT-Labinstruments GmbH, Salzburg, Austria) at 415 nm.Standard curve was produced with commercial β-Gal (Sigma).

[0083] IL-2 production assay: IL-2 was assayed according to theestablished bioassay procedure (Mosmann, 1983). CTL-L cells were washed3 times in medium and resuspended at 2×10⁵ cells/ml. 50 μl of cells wereplaced in a well of a 96-well plate in the presence of 50 μl of thesupernatant to be assayed. Following 20 hours incubation, 10 μl of 5mg/ml MTT (Sigma) was added and the plates were incubated for additional4 hours. 100 μl of isopropanol containing 0.04 N HCl was added, and thereactions were thoroughly mixed. Plates were read at 570 nm with 620 nmas reference in SLT Spectra ELISA Reader.

Example 1 Design of an MHC Class I Model System

[0084] In order to examine the feasibility of the conception of theinvention, a class I MHC murine model system was mounted according tothe general scheme presented in FIG. 1. As the MHC component,monomorphic human β₂m was chosen. It associates with all class I heavychains, thus allowing universality of use, both experimentally andtherapeutically. As the signaling domain, the transmembranal andcytoplasmic portions of the mouse CD3 ζ chain was chosen.

[0085] As the effector cells we used MD45 cells, a CD8⁻H-2^(b)-alloreactive H-2^(d/k) mouse CTL hybridoma (Kaufmann, et al.,1981). These cells grow readily in culture, are highly transfectable andsecrete considerable amount of cytokines following activation. Theirlytic capacity is, however, partially compromised, compared with primaryCTLs. It was chosen to work with human rather than mouse β₂m in thesecells, as it provides a distinct marker for the product of the chimericgene. Human β₂m is known to pair efficiently with most mouse class Iheavy chains, and it does so especially well with the K^(k) molecule(Schmidt et al., 1981). To assure optimal interactions between thesignaling domains and downstream membranal and cytoplasmic components ofmouse origin involved in the signal transduction pathway, it was chosento use mouse rather than human ζ chain.

[0086] For assessment of peptide-specific responses, which are relevantto autoimmunity, it was chosen to express the antigenic peptides linkedto the amino terminus of the chimeric P₂m molecule via a flexiblepeptide linker. This mode of expression allows the generation of stablytransfected hybridoma cells which constitutively present the antigenicpeptides on their class I molecules.

Example 2 Class I MHC Construct Containing hβ₂m

[0087] Human β₂m (hβ₂m) cDNA was cloned by RT-PCR, using mRNA preparedfrom Jurkat (human T cell leukemia) cells, with the following primers:1st strand primer, 4337, containing an XhoI restriction site: 5′ G CTGGCT CGA GGG CTC CCA TCT CAG CAT GTC TCG ATC CCA CTT 3′ 2nd strandprimer, 30287, containing an XbaI site: 5′ GGG TCT AGA GCC GAG ATG TCTCGC TCC GTG 3′

[0088] A cDNA segment encoding the transmembranal and cytoplasmicregions of the murine CD3 ζ chain was cloned by RT-PCR from mRNA of MD45cells with the following primers: 1st strand primer, 27246, containingan EcoRI site: 5′ GCG GAA TTC TTA GCG AGG GGC CAG GGT 3′ 2nd strandprimer, 4840, containing an XhoI site: 5′ GAG CCC TCG AGC CAG CCC ACCATC CCC ATC CTC TGC TAC TTG CTA GAT 3′

[0089] While associated with the class I heavy chain HLA-A2, thedistance of the C terminus of hβ₂m from the cell membrane is equivalentto that occupied by the 13 membrane-proximal amino acids (Bjorkman, etal., 1987) of the heavy chain. In an attempt to preserve class I spatialorganization, this HLA-A2-derived 13-amino acid stretch was chosen tolink hβ₂m to the membrane. This sequence has been incorporated into thecloned genes via the PCR primers 4337 and 4840. The restriction sitesincluded in the PCR products have been used for a single step insertionof both products into the pBJ1-Neo expression vector (Lin et al., 1990),cleaved with XbaI+EcoRI, to produce clone 21-2. The resulting chimericgene codes for hβ₂m, bridged to the cell and equipped with 4 chainsignal transduction domains, as illustrated in FIG. 2.

[0090] The assembled gene comprises the following DNA stretches:

[0091] 1. hβ₂m GenBank Accession (G.B.A.) AF072097, positions 801-1157

[0092] 2. HLA-A2 G.B.A. K02883: 2380-2389+2489-2517. This sequence isCTG AGA TGG GAG CCG TCT TCC CAG CCC ACC ATC CCC ATC and it encodes the13-amino acid peptide: Leu Arg Trp Glu Pro Ser Ser Gln Pro Thr Ile ProIle. The original Pro-Ser-Ser coding stretch was converted into CCC TCGAGC, encoding the same amino acids, so as to create an XhoI cloning site(CTCGAG).

[0093] 3. Mouse CD3 ζ transmembrane (tm)+cytoplasmic (cyt) G.B.A.M19729: 193-597. The corresponding sequence in the human ζ tm+cyt isG.B.A. J04132: 165-566.

[0094] The complete nucleotide sequence assembled coding region and itshuman β₂m and mouse CD3 ζ chain gene constituents are presented ini FIG.3.

[0095] This DNA construct was transfected into MD45 cells.G418-resistant clones were subjected to FACS analysis, using ananti-hβ₂m mAb (Sigma M7398, St Louis, Mo., USA), in order to evaluatecell surface expression of the chimeric polypeptide. Approximately 50%of the clones were intensively stained. Staining of representativeclones 29 and 49 are shown in FIG. 4. To test whether the chimeric β₂mcreates functional MHC complexes on the cell surface, plastic wells werecoated with a rat anti-mouse anti-H-2K^(k) mAb (Zymed 24-1400, SanFrancisco, Calif., USA)), and transfectants were assayed for theirability to secrete interleukin 2 (IL-2) following incubation with theimmobilized antibody. Indeed, 2 of the clones tested, 29 and 49,secreted significant amount of the lymphokine to the medium, asmonitored by a CTL-L bioassay. These have been subcloned (clones 29-2and 49-10) and a high level of secretion was now evident, as shown inFIG. 5. These results demonstrate both functional association of thechimeric hβ₂m with the K^(k) heavy chain and coupling of theseengineered class I molecule to the T-cell activation pathway.

Example 3 Construction of a Reporter System

[0096] Specific interactions between engineered effector cells andtarget T cells is expected to result in cytokine release by bothparties. If both are of mouse origin, the source of the detectedcytokines will be ambiguous. It was chosen to tackle this problem byco-transfecting the MD45 cells with plasmid 21-2 obtained in Example 2above and with a genetic construct encoding the lacZ reporter genedriven by the minimal promoter of the human IL-2 gene [NF-AT-LacZ,kindly provided by Dr. N. Shastri, University of California (Karttunenet al., 1991)]. This was transfection 412. In addition, MD45 cells wereco-transfected with the NF-AT-lacZ reporter gene and the pBJ1-Neo vector(transfection 392). For preliminary screening, G418-resistant cloneswere incubated with the T-cell mitogen Con A, and IL-2 gene activationwas evaluated by the colorimetric ONPG assay, which monitors β-Galactivity. ONPG detection assay was again used with the strongest clonesfrom each transfection incubated with: 1) Con A; 2) an anti-H-2K^(k) mAb(Pharmingen A6051D); 3) no stimulation. Results of clones and MD45parental cells are presented in Table 1. These results indicate thatthat the reporter gene is functional in the transfected cells and thatthe chimeric β₂m molecule recruits NF-AT, the T-cell specific nuclearfactor, as a downstream transcription factor in the TCR-mediatedsignaling pathway. Clones 412-19 and 392-14 were further subcloned,giving rise to clones 412-19-1-6 and 392-14-1, respectively.

[0097] β-Gal enzymatic assay was carried out with MN45 parentalhybridoma cells, a transfectant expressing both the chimeric hβ2m/ζconstruct and the NF-AT-LacZ reporter gene (412-19) and anothertransfectant (392-14), expressing only the reporter gene, were assayed.Results from a representative experiment are presented in Table 1 asOD₄₀₅ of a calorimetric Lac-Z assay. TABLE 1 Clone Con A Anti-H-2 K^(k)No Stim. 412-19 0.074 0.160 0.002 392-14 0.395 0.007 0.011 MD45 0.001

Example 4 Assembling Genetic Constructs for Testing Peptide Specificity

[0098] In order to target alloreactive T cells, the chimeric β₂m/ζ chainconstruct may be sufficient. However, since autoreactive T cells usuallyrecognize a specific self-peptide in the context of MHC, it wasimportant to demonstrate that the effector cells can respond toK^(k)-restricted CD8⁺ T cells specific to a given peptide, if the latteris presented by the transfectants' K^(k). To perform experiments alongthis line we exploited two existing K^(k)-restricted murine CD8⁺ T cellhybridomas [kindly provided by Dr. A. Stryhn, University of Copenhagen(Stryhn et al., 1994)]. One hybridoma, HK9.5-24, is specific to aninfluenza virus nucleoprotein (NP) peptide (NP50-57), and the other,HK8.3-5H3, is specific to an influenza virus hemagglutinin (Ha) peptide(Ha255-262). These hybridomas and peptides were only employed as a modelsystem for peptide specificity, which provides solid internal controls,and have no biological relevance to, and are not encompassed by, thepresent invention.

[0099] In addition, we chose to study the insulin B chain (IB) peptide,associated with K^(d). This setting is relevant to the diabetes animalmodel in NOD mice, and is designed to be tested with theK^(d)-restricted, diabetogenic CD8⁺ clone G9C8, specific to this peptide(Wong, et al., 1999).

[0100] In order to obtain high density of peptides NP50-57 and Ha255-26and the peptide from insulin B chain (IB) on the effector T cells of thepresent invention, three genetic constructs were assembled designed tolink those peptides to the amino terminus of hβ₂m covalently via aflexible peptide linker (see FIG. 6A). This configuration has beenrecently shown to create functional CTL targets with H-2 D^(b), K^(d)and D^(d) (Uger et al., 1998; Uger et al., 1999; White et al., 1999).The genetic design of these constructs is schematically delineated inFIG. 6B. The linker peptide chosen for this purpose was the one used byWhite et al., 1999, namely, a 13-amino acid peptide of the sequence:Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser Gly-Gly-Gly-Ser, encoded by: GGA GGTGGC GGG TCC GGA GGT GGT TCT GGT GGA GGT TCG.

[0101] We have changed the last G in the 4th Gly to an A, thus creatinga unique BamHI site (GGATCC), which was useful in the cloning procedure.

[0102] 4a. Genetic Construct Containing the Peptide NP50-57

[0103] The 5′ primer of the upstream fragment was 30287 (see Example 2above) and was the same for all 3 peptides, corresponding to the 5′ endof hβ₂m leader peptide, and harboring an XbaI site. The original NP50-57amino acid sequence is:

[0104] Ser Asp Tyr Glu Gly Arg Leu Ile.

[0105] The NP nucleotide sequence of A/Japan/305/57 could not be foundin the gene bank, and the nucleotide sequence was derived from a relatedstrain, H3N2 A/Akita/1/94, G.B.A. U71144: 148-171: AGT GAT TAT GAA GGGCOGG TTG ATC.

[0106] The 3′ primer was 6086, containing a BamHI restriction site:5′ CGC GGA TCC GCC ACC TCC GAT CAA CCG CCC TTC ATA ATC ACT AGC CTC AAGGCC AGA AAG 3′

[0107] The 2nd set of primers was designed to amplify the rest of thelinker and the fill, mature hβ₂m with the part of the HLA-A2 bridge.

[0108] The 5′ primer, 5187, was the same for the 3 peptides and containsa BamHI site: 5′ GCG GGA TCC GGA GGT GGT TCT GGT GGA GGT TCG ATC CAG CGTACT CCA AAG 3′

[0109] The 3′ primer for all peptides was 4337 (see Example 2 above)containing an XhoI site.

[0110] 4b. Genetic Construct Containing the Peptide Ha 255-262

[0111] The original Ha 255-262 peptide of influenza virus strainA/Japan/305/57 has the sequence: Phe Glu Ser Thr Gly Asn Leu Ile, and isencoded by G.B.A. L20407: 806-829 TTT GAG AGT ACT GGT AAT CTA ATT.

[0112] The 3′ primer was 6087, with a BamHI restriction site: 5′ CGC GGATCC GCC ACC TCC AAT TAG ATT ACC AGT ACT CTC AAA AGC CTC AAG GCC AGA AAG3′

[0113] 4c. Genetic Construct Containing the IB Peptide

[0114] The peptide sequence is Leu Tyr Leu Val Cys Gly Glu Arg Gly, andit is derived from mouse preproinsulin: G.B.A. X04725: 943-969.

[0115] CTC TACGCTG GTG TGT GGG GAG CGT GGC

[0116] The 3′ primer is 6338, with a BamHI restriction site: 5′ CGCGGA TCC GCC ACC TCC GCC ACG CTC CCC ACA CAC CAG GTA GAG AGC CTC AAG GCCAGA AAG 3′

[0117] The 3 final constructs were assembled in one cloning step. TheXbaI/BamHI fragment for each of the 3 peptides (following subcloning andnucleotide sequence determination) was joined with the BamHI/XhoIfragment (the same one for all peptides—also subcloned and sequenced)and with the Xho/EcoRI fragment from clone 21-2, to the pBJ1-Neo vectorcleaved with XbaI+EcoRI. The resulting clones were 406-20 for the NPpeptide, 407-4 for the Ha peptide and 408-9 for the IB peptide. The DNAsequence of their coding stretches is presented in FIG. 7.

Example 5 Analysis of the Expression of the Chimeric β₂m with Peptides

[0118] The three plasmids of Examples 4a, 4b and 4c above were eachco-transfected into MD45 cells together with the NF-AT-lacZ reportergene. Clones generated with plasmid 406-20 were designated 797, thosegenerated with plasmid 407-4 were designated 798, and plasmid 408-9yielded clones 799. Twenty four G418 resistant clones from eachtransfection were tested for IL-2 secretion following incubation withimmobilized anti-H-2K^(k) mAb for NP and Ha peptides or anti-H-2K^(d)mAb (Pharmingen 06091D) for the insulin peptide. Stimulation wasmonitored by an IL-2 bioassay, using the IL-2-dependent CTL-L cell line.IL-2 production was assayed after stimulation of MD45 cells transfectedwith chimeric antigenic peptide/hβ₂m/ζ genes by an immobilizedanti-H-2K^(k) or anti-H-2K^(d) mAb. IL-2 was monitored with a CTL-Lbioassay, which was developed via an M4TT calorimetric assay (see“Materials and Methods”). MD45 cells serve as a negative control.Results are shown in Table 2. 797, 798, 799—are transfectants with NP-,Ha- and IB-encoding chimeric genes, respectively; clone 412-412-19-1-6,serves as a positive control; TABLE 2 Clone O.D. Clone O.D. Clone O.D.797 (anti-K^(k)): 798 (anti-K^(k)): 799 (anti-K^(k)): 1 0.007 1 0.011 10.006 2 0.003 2 0.078* 2 0.002 3 0.162** 3 0.013 3 0.012 4 0.003 4 0.0094 0.143** 5 0.001 5 0.008 5 0.159** 6 0.002 6 0.007 6 0.005 7 0.007 70.011 7 0.005 8 0.004 8 0.008 8 0.166** 9 0.024* 9 0.048* 9 0.007 100.161** 10 0.008 10 0.158** 11 0.008 11 0.005 11 0.026* 12 0.005 120.006 12 0.026* 13 0.008 13 0.011 13 0.022* 14 0.007 14 0.031* 15 0.01015 0.009 15 0.149** 16 0.039* 16 0.069* 16 0.029* 17 0.004 17 0.019 170.100** 18 0.113** 18 0.082* 18 0.091* 19 0.006 19 0.052* 19 0.055* 200.020* 20 0.006 20 0.013 21 0.011 21 0.120** 21 0.154* 22 0.020* 220.010 22 0.012 23 0.008 23 0.000 23 0.008 24 0.050* 24 0.006 MD45 0.003MD45 0.004 412 0.171** 412 0.161**

[0119] These results lend further support to our previous findings thatthe chimeric β₂m functionally associates on the cell surface withendogenous class I H chains, and adds H-2K^(d) to H-2K^(k) as moleculeswhich are capable of transducing T cell activation signals.Unfortunately, none of the transfected clones which produced high levelof IL-2 in response to the immobilized mAbs showed significant β-Galactivity following stimulation.

[0120] We decided to proceed by following a step-wise transfectionprotocol. This time, MD45 cells were transfected only with the chimericgene. Cells transfected with plasmid 406-20 encoding the NP peptidegenerated series 419 of clones. Similarly, MD45 transfected with plasmid407-4 encoding the Ha peptide resulted in clones denoted 420. In bothcases selection was carried out with G418.

[0121] Table 3 summarizes the results of an IL-2 bioassay of a screeningexperiment of these two series of clones, showing all clones whichelicited significant IL-2 production above background with any of thetwo K^(k)-restricted CTL hybridomas described above—hybridoma HK9.5-24specific to NP50-57 and hybridoma K8.3-5H3 specific to Ha255-262. Allclones were incubated in this experiment simultaneously with the twohybridomas in exactly the same conditions, as described in “Materialsand Methods”. This setting allows cross-reference of the tested clones,thus constituting a reliable internal control.

[0122] IL-2 production was assayed after co-incubation of clonestransfected with the NP-encoding chimeric gene (419 series) or with theHa-encoding gene (420), with the two target cells. IL-2 was monitoredwith the CTL-L bioassay. Only transfected cells which inducedsignificant IL-2 production have been included. Note that under thisexperimental setting it is impossible to identify the source of thesecreted lymphokine. TABLE 3 Clone HK8.3-5H3 (Ha) HK9.5-24 (NP) NP:419-6  0.030 0.068 419-17 0.007 0.101 419-19 0.013 0.058 419-21 0.0150.032 419-27 0.007 0.036 419-28 0.011 0.029 419-40 0.023 0.105 419-490.019 0.045 419-55 0.006 0.050 419-60 0.019 0.060 419-61 0.053 0.121419-65 0.011 0.062 Average 0.018 0.064 Ha: 420-11 0.053 0.012 420-270.031 0.018 420-31 0.030 0.014 420-35 0.038 0.016 420-40 0.037 0.011420-44 0.029 0.016 420-52 0.039 0.016 420-58 0.040 0.012 420-62 0.0360.012 420-66 0.053 0.019 420-71 0.025 0.007 Average 0.037 0.014

[0123] This experiment indicates that co-incubation of MD45transfectants containing a construct according to the inventionexpressing the influenza NP peptide with both K^(k)-restricted CTLhybridomas, results in significantly higher IL-2 secretion wit HK9.5-24.The inverse is true with cells expressing the Ha peptide, which inducemuch higher IL-2 production when incubated with HK8.3-5H3 cells. We wereunable to detect a clone exhibiting the opposite profile of response.The results strongly indicate that the chimeric antigenic peptide-β₂m-ζchain polypeptide is properly expressed on the cell membrane inassociation with K^(k) H chain. It should be noted that theseexperiments are not designed to determine the source of the secretedcytokine. At this stage, however, it is clear that a peptide-specificresponse does indeed take place following co-incubation of our modifiedT cells with their target CTL hybridomas, thus, that the peptide ispresented on the cell surface on K^(k) in a constitutive manner.

Example 6 Assay of Peptide Specificity of Transfected T Cells

[0124] As discussed above, demonstrating peptide-specific response ofour clones requires unambiguous assignment of specific response to themodified MD45 effector cells, a challenge we chose to tackle by the useof the reporter gene. Implementing the approach of stepwisetransfections, we chose clones 419-17 and 420-11 as the best responders,and super-transfected them with the NF-AT-LacZ reporter gene, togetherwith an additional plasmid pMCC-ZP (kindly provided by Dr. J.Lazarowitz, Bio-Technology General, Rehovot, Israel) which allows forpuromycin selection. Clones resistant to both G418 and puromycin werefirst tested by a β-Gal enzymatic assay for their ability to produce theenzyme in response to Con A stimulation. Cell lysates of two clonesoriginating from 419-17, numbered 425-44 and 425-68, showed strongenzymatic activity following incubation in the presence of the Con A.One of these clones, 425-68, was then assayed for β-Gal productionfollowing stimulation by a serial dilution of the immobilized anti-K^(k)mAb Pharmingen): A dose-dependent response was obtained and is presentedin FIG. 8.

[0125] These two clones were then assayed (as described in “Materialsand Methods”) with each of the two CTL hybridomas 9.5-24 specific toNP50-57 and K8.3-5H3 specific to Ha255-26, with Con A as a positivecontrol and with no stimulation as a negative control, monitoring basalβ-Gal production by these cells. The results for clone 42544 are shownin FIG. 9. These results were typical to those obtained in similarexperiments performed with the full set of two effectors and twotargets, as described below, and demonstrated the expectedpeptide-specificity of the chimeric MHC-mediated activation.

[0126] We could not identify any strong β-Gal producers among thedescendants of clone 420-11. We then decided to implement our stepwisetransfection procedure in an inverted order. We started with clone392-14-1 (see above) which expresses high level of β-Gal uponstimulation (and is G418-resistant) and introduced into these cellsplasmid 407-4 (encoding the Ha peptide), together with pMCC-ZP to conferpuromycin resistance. One double-resistant clone generated in thistransfection, 427-24, displayed the expected phenotype. It producedβ-Gal following incubation with immobilized anti-H-2K^(k) mAb, and itled to IL-2 secretion after co-incubation with the Ha peptide-specificCTL hybridoma, HK8.3-5H3.

[0127] We then performed a series of co-incubations of our clones 425-44and 427-24 with both target CTL hybridomas, assaying β-Gal activity, allas described in “Materials and Methods”. Results are summarized in FIG.9. Although the magnitude of the observed response in this type of assayis relatively low, its anticipated profile, namely, correlation betweenthe antigenic peptide expressed by the responding clone and theantigenic specificity of the target CTL hybridoma, is clearly evident,as supported by the high significance value. We attribute theserelatively low values to a number of factors. First, MD45 cells andtheir descendant clones express significantly lower levels of K^(k), ascompared to normal cells, as observed with a fluorescent microscope,using the anti-^(k) mAb (data not shown). Second, as reported before(Stryhn et al., 1994) and confirmed by us, the hybridoma target cells donot express CD8. Third, MD45 are negative for both CD8 and CD4.Thesedeficiencies are likely to decrease the actual number of functionalinteractions between the engineered MHC molecules on the effector cellsand TCRs on the target cells, reduce their avidity and affect themagnitude of the transduced signal.

[0128] The results obtained above in our MSC class I model cell systemdemonstrates that MHC class I molecules can be converted into T cellactivation receptors, and that T cells expressing these receptors can beactivated in a peptide-specific manner.

Example 7 Construction of a Class II MHC Model System

[0129] Antigen presentation by class II MHC molecules on the cellsurface requires a number of accessory molecules. The more importantones are a protein called invariant (Ii) chain, which acts as a chaperonfor the newly synthesized class II molecules, and a class II 1HC-likemolecule called HLA-DM (in humans) or H-2M (in mice), which catalyzesloading of class II molecules with endogenously-processed peptides.These proteins are not normally expressed by cells which do not expressclass II, such as mouse T cells. Unlike, activated human T cells (bothCD4⁺ and CD8⁺) do express surface class II HLA and are known to functionas effective antigen-presenting cells (Barnaba et al., 1994).

[0130] To test chimeric HLA class II expression and its function in ahuman T cell system, we chose HLA-DR2, which is highly associated withMS. Several DR2 alleles have been identified, among which DR2Dw2 is themost common in DR2⁺ individuals and in most DR2⁺ MS patients(Wucherpfennig et al., 1991). Its α chain (as for all DR molecules) isencoded by the DRA*0101 gene and its β chain by DRB1*1501. The sourcefor both these genes was an EBV-transformed human B cell line (kindlyprovided by Dr. D. Teitelbaum, The Weizmann Institute of Science,Rehovot, Israel). mRNA prepared from these cells served for RT-PCRamplification of the desired fragments.

[0131] 7a. Assembly of DRα/Mouse ζ Chain Construct

[0132] A chimeric gene encoding the extracellular part of DRα joined tothe transmembranal and cytoplasmic domains of mouse ζ chain wasassembled from two components:

[0133] 1. An 0.65 kb XhoI/XbaI fragment containing the 5′ end ofHLA-DRA*0101, including the segment encoding the leader peptide, joinedto the 5′ end of the stretch encoding the mouse ζ chain transmembranaldomain.

[0134] The 5′ primer, 32152, contains an XhoI restriction site and isspecific to DRA*0101 from position 11 in GenBank Accession J00194,including the ATG initiation codon.

[0135] 5′ GGC CCC TCG AGG CGC CCA AGA AGA AAA TGG CC 3′

[0136] The 3′ primer, 3982, contains an XbaI site. It allows thein-frame joining of the 3′ end of the stretch encoding the DRαextracellular portion (up to position 635 in the gene) with the 5α endof the transmembranal domain of mouse ζ (from position 193 in GenBankAccession M19729). The XbaI restriction site was introduced into the ζchain coding sequence without changing the amino acid composition.

[0137] 5′ GGG TCT AGA AGG TAG CAG AGC CAC TGC TTG AGA AGA GGC 3′

[0138] 2. An 0.4 kb XbaI/EcoRI fragment encoding the ζ chaintransmembrane and cytoplasmic domain.

[0139] The 5′ primer 25528 is specific for the 5′ of the ζ domain (seeExample 1 above) and contains an XbaI site:

[0140] 5′ GGG TCT AGA TGG AAT CCT CTT CAT C 3′

[0141] The 3′ primer is the primer 27246 used in Example 1 above for theassembly of the chimeric β₂m gene.

[0142] Both PCR products were cloned in a single step into theexpression vector pBJ1-Neo prepared with XhoI+EcoRI, to produce clone23-2. It is schematically presented in FIG. 10A and the completenucleotide sequence of its coding region is shown in FIG. 10B.

[0143] 7b. Cloning DRB*1501

[0144] The DRB*1501 gene encoding the DRβ chain was cloned in its nativeform, including the leader sequence, as a single PCR product, using thefollowing primers:

[0145] The 5′ one, 28239, harbors an XhoI site, and is specific topositions 17-34 in GenBank Accession M20430. 5′ CGC GCC TCG AGC CCC TGGTCC TGT CCT G 3′ The 3′ primer, 201793, is specific to the 3′ end of thegene (positions 1114-1133): 5′ GTA ATG TGT TTG TCA TAC AG 3′

[0146] This PCR product was then cleaved with XhoI and HindIII (locatedapproximately. 60 bp downstream of the gene's stop codon of theDRB1*1501 gene), and inserted into pBJ1-Neo.

[0147] The chimeric DRα/ζ and the native DRβ genes can be expressed in asuitable host cell such as, for example, in human T cells immortalizedby Herpes virus saimiri [H. saimiri, reviewed by Meinl et al., 1995).This lymphotropic agent has been shown to transform human CD4⁺ and CD8⁺T cells to continuous growth in culture for extended periods,independently of stimulation with APC and antigen. HLA-DR-restricted H.saimiri-transformed CD4⁺ T cell clones (Weber et al., 1993) can alsoserve as specific targets in this system. MBP-specific lymphokinesecretion and cytolysis by the CD8⁺ cells can be assayed in experimentsanalogous to those described for the class I system. Efficient class IIpresentation of MBP peptides by the chimeric DR/ζ transfectants isexpected following their incubation with MBP or its reelevant peptides(Barnaba, et al., 1994). Other MS- and diabetes-related autoantigens andadditional HLA-DR restricting elements can be tested by the sameexperimental system.

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1. A DNA molecule encoding a chimeric polypeptide comprising (a) acomponent of a MHC molecule capable of association on a cell surfacewith an endogenous MHC molecule component of the same class, and (b) anintracellular region of a signal transduction element capable ofactivating T cells.
 2. A DNA molecule of claim 1, wherein said MHCcomponent (a) is a monomorphic component selected from β₂-microglobulinand the monomorphic α chain of a HLA-DR molecule.
 3. A DNA molecule ofclaim 2, wherein said MHC component (a) is human β₂-microglobulincapable of association on a cell surface with an endogenous class Iheavy chain HLA molecule, and said β₂-microglobulin is linked tocomponent (b) by a bridge peptide having about 10-15 amino acidresidues.
 3. A DNA molecule of claim 2, wherein said bridge peptide hasa sequence comprised within the membrane-proximal sequence of a class Iheavy chain HLA molecule.
 4. A DNA molecule of claim 2 or 3, whereinsaid bridge peptide has 13 amino acid residues comprised within theextracellular membrane-proximal sequence of the class I heavy chainHLA-A2 molecule, and has the sequence: Leu Arg Trp Glu Pro Ser Ser GlnPro Thr Ile Pro Ile
 5. A DNA molecule of claim 2, wherein said MHCcomponent (a) is a monomorphic α chain of a HLA-DR molecule.
 6. A DNAmolecule of claim 1, wherein said MHC component (a) is a polymorphiccomponent selected from: (i) an α chain of class I HLA-A, BLA-B or HLA-Cmolecule, which is capable of association on a cell surface withendogenous β₂m; (ii) an α chain of class II HLA-DP or HLA-DQ molecule,which is capable of association on a cell surface with endogenousHLA-DPβ or HLA-DQβ chain; or (iii) a β chain of class II HLA-DP, HLA-DRor HLA-DQ molecule, which is capable of association on a cell surfacewith an endogenous HLA-DPα, HLA-DRα or HLA-DQα chain.
 7. A DNA moleculeof any one of claims 1 to claim 6, wherein said intracellular region (b)of a signal transduction element capable of activating T cells isselected from a component of T-cell receptor CD3, a B cell receptorpolypeptide or an Fc receptor polypeptide.
 8. A DNA molecule of claim 7,wherein said component of T-cell receptor CD3 is the zeta (ζ) or eta (η)polypeptide.
 9. A DNA molecule of claim 8, wherein said component ofT-cell receptor CD3 comprises the transmembranal and cytoplasmic regionsof the human ζ polypeptide.
 10. A DNA molecule of any one of claims 1 to9, wherein said chimeric polypeptide further comprises an antigenicpeptide related to an autoimmune disease, said antigenic peptide beinglinked to said chimeric polypeptide by a peptide linker.
 11. A DNAmolecule of claim 10, wherein said antigenic peptide has 8-10 amino acidresidues and binds to a product of a certain HLA allele.
 12. A vectorcomprising a DNA molecule of any one of claims 1 to
 11. 13. A cell whichexpresses a chimeric polypeptide comprising (a) a component of a MHCmolecule capable of association on a cell surface with an endogenous MHCmolecule component of the same class, and (b) an intracellular region ofa signal transduction element capable of activating T cells.
 14. A cellof claim 13, wherein said cell is an immune cell selected from T helpercells (CD4⁺), cytotoxic T lymphocytes (CD8⁺) and natural killer (NK)cells, capable of recognizing and binding to harmful T cells and causingtheir elimination or inactivation.
 15. An immune cell which expresses achimeric polypeptide as defined in any one of claims 1 to 9 and bindsto, and eliminates, alloreactive cells causing transplant rejection. 16.An immune cell of claim 15 which is a cytotoxic T lymphocyte (CTL). 17.An immune cell which expresses a chimeric polypeptide as defined inclaim 10 or 11 and binds to and eliminates autoreactive cells causing anautoimmune disease.
 18. A method for the prevention and/or treatment ofgraft-versus-host disease (GVHD) which comprises administering to apatient in need thereof at suitable times autologous T cells whichexpress a chimeric polypeptide comprising (a) a component of a MHCmolecule capable of association on a cell surface with an endogenous MHCmolecule component of the same class, and (b) an intracellular region ofa signal transduction element capable of activating T cells.
 19. Amethod for the prevention and/or treatment of host-versus-graft reactionwhich comprises administering to a patient in need thereof at suitabletimes donor T cells which express a chimeric polypeptide comprising (a)a component of a MHC molecule capable of association on a cell surfacewith an endogenous MHC molecule component of the same class, and (b) anintracellular region of a signal transduction element capable ofactivating T cells.
 20. A method for the prevention and/or treatment ofan autoimmune disease which comprises administering to a patient in needthereof autologous T cells which express a chimeric polypeptidecomprising an antigenic peptide related to said autoimmune diseaselinked by a peptide linker to a component of a MHC molecule capable ofassociation on a cell surface with an endogenous MHC molecule componentof the same class, followed by an intracellular region of a signaltransduction element capable of activating T cells.
 21. A method for theprevention and/or treatment of an autoimmune disease which comprisesadministering to a patient in need thereof autologous T cells whichexpress a chimeric polypeptide comprising (a) a component of a MHCmolecule capable of association on a cell surface with an endogenous MHCmolecule component of the same class, and (b) an intracellular region ofa signal transduction element capable of activating T cells, and whereinone or more antigenic peptides related to said autoimmune disease areexogenously loaded in the grooves of the MHC complex formed by theassociation of component (a) with the endogenous MHC molecule component.22. A method according to claim 21 wherein said one or more antigenicpeptide are exogenously supplied by incubation of the cells with one ormore peptides or proteins associated with said autoimmune disease.